All of the manifestations of alphaherpesvirus disease (herpes simplex, varicella zoster) result from the ability of the virus to spread from the initial infected cell or cells at mucosal surfaces to other cells in the area and to nerve cells that innervate the site of primary replication. Recurrence of symptoms and consequent spread of the virus to new hosts similarly requires the ability to spread from the sensory ganglion cells to cell at the periphery and among the cells on the mucosal surface. Amazingly, spread of the virus in recurrent infection occurs in the face of an adaptive immune response, including an antibody response that should neutralize virus released from the cell. The disease-causing properties of these viruses therefore depend on the mechanisms used for spread from cell to cell that protect the virus from exposure to effectors of the adaptive immune response. Spread of the human alphaherpesviruses within the host requires trafficking of newly assembled virus particles from their assembly site at the Golgi to exposed cell surfaces for release to extracellular medium or to cell junctions for cell-to-cell spread (CCS). Neither trafficking pathway is well understood. In part this is because, other than viral proteins required for entry of virus into host cells, no virl gene functions have been identified that are required for the process in most cell types. We have discovered that two viral gene products, pUL34 and pUL51, play critical roles in efficient virus release and/or CCS. Both proteins are apparently multifunctional. pUL34 is required for nuclear egress of herpesvirus capsids, and pUL51 has been shown to be required for efficient cytoplasmic assembly of the virus. We have discovered, however, that both proteins play critical roles in release and CCS that can be genetically uncoupled from their roles in virion assembly. Our overall goal is to test the hypothesis that HSV release and CCS are accomplished by viral hijacking of cellular pathways that sort host cell membrane proteins to appropriate surfaces and junctions. We will use two general approaches to this overall goal. The first approach (contained in the first two specific aims) is to characterize the functions and interactions of pUL34 and pUL51 that are required for virus release and CCS. These viral proteins can thus be used as tools to identify critical viral and cellular proteins that also participate. The second approach (contained in the third specific aim is to take advantage of recently developed information about the host pathways that deliver cellular membrane proteins to basolateral and junctional surfaces of cells and to probe those pathways using dominant negative inhibitors of critical molecules.